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The recent appearance and worldwide spread of novel influenza A (H1N1) virus (1,2) has highlighted the need to evaluate commercially available, widely used, rapid influenza diagnostic tests (RIDTs) for their ability to detect these viral antigens in respiratory clinical specimens. As an initial assessment, CDC conducted an evaluation of multiple RIDTs. Sixty-five clinical respiratory specimens collected during April--May 2009* that had previously tested positive either for novel influenza A (H1N1) or for seasonal influenza A (H1N1) or A (H3N2) viruses by real-time reverse transcription--polymerase chain reaction (rRT-PCR) assay were used in the evaluation. The results showed that, although the RIDTs were capable of detecting novel A (H1N1) virus from respiratory specimens containing high levels of virus (as indicated by low cycle threshold [Ct] values), the overall sensitivity was low (40%--69%) among all specimens tested and declined substantially as virus levels decreased (and Ct values increased). These findings indicate that, although a positive RIDT result can be used in making treatment decisions, a negative result does not rule out infection with novel influenza A (H1N1) virus. Patients with illnesses compatible with novel influenza A (H1N1) virus infection but with negative RIDT results should be treated empirically based on the level of clinical suspicion, underlying medical conditions, severity of illness, and risk for complications. If a more definitive determination of infection with influenza virus is required, testing with rRT-PCR or virus isolation should be performed. Additional evaluations of the accuracy of RIDTs in detecting novel influenza A (H1N1) virus should be conducted.

Original clinical materials (e.g., specimens from nasopharyngeal swabs and oropharyngeal swabs) collected from patients with confirmed novel influenza A (H1N1) or seasonal influenza A (H1N1) or (H3N2) virus infection and provided largely by state health laboratories were used in the study. The presence of novel or seasonal influenza A virus was confirmed by rRT-PCR assay developed by CDC and approved as a Section 501(k) device by the Food and Drug Administration. Detailed data regarding sensitivity (99.3%) and specificity (92.3%) for the seasonal influenza A CDC rRT-PCR assay compared with viral culture are available.† The original clinical specimens were tested using RIDTs from three companies: Inverness Medical BinaxNOW Influenza A&B (Binax, Inc., Scarborough, Maine); Becton Dickinson Directigen EZ Flu A+B (Becton, Dickinson and Company, Sparks, Maryland); and Quidel QuickVue Influenza A+B (Quidel Corporation, San Diego, California). RIDTs from four other companies were tested with limited numbers of specimens; those results are not presented in this report.

Each clinical specimen was characterized by the Ct value demonstrated in the universal influenza type A rRT-PCR assay with the M gene used as the target.§ The numbers of specimens positive using each of the three RIDTs were determined within four intervals of Ct values: <20, 20 to <25, 25--30, and >30.¶ Ct values are indicators of the amount of virus in a specimen, with lower values indicating higher viral titers (i.e., greater amounts of viral material in the specimen). Sensitivity of each rapid test was determined as the percentage of RIDT-positive specimens among the number of specimens that tested positive by rRT-PCR.

A total of 65 original clinical specimens were tested. Forty-five of the specimens were positive for novel influenza A (H1N1) virus, five were positive for seasonal influenza A (H1N1), and 15 were positive for seasonal influenza A (H3N2), all by CDC rRT-PCR assay.

For the nine specimens with high viral titers (Ct values <20), one RIDT had nine positive results, and the other two had eight positives, demonstrating 89%--100% sensitivity in detecting novel influenza A (H1N1) virus when compared with rRT-PCR. However, among the 36 specimens with Ct values ≥20 that had tested positive for novel influenza A (H1N1) by rRT-PCR, the sensitivity of the three RIDT tests declined substantially (Table 1). Overall, for the 45 specimens that had tested positive for novel influenza A (H1N1) by rRT-PCR, the sensitivity of the three RIDT tests was 40% for BinaxNOW Influenza A&B, 49% for Directigen EZ Flu A+B,** and 69% for QuickVue Influenza A+B.

Sensitivity of the RIDTs was generally greater for seasonal influenza A (H1N1) and (H3N2) than for novel influenza A (H1N1), although the number of specimens tested was small, especially for seasonal influenza A (H1N1). None of the specimens had a Ct value <20. Compared with rRT-PCR, the three tests demonstrated sensitivity ranging from 60% to 80% for seasonal A (H1N1) and from 80% to 83% for seasonal A (H3N2) (Table 1).

To evaluate approximate viral titers in clinical specimens positive for novel influenza A (H1N1) virus, serial 10-fold dilutions (from 10-1 through 10-5) of the virus isolate A/California/4/2009, an early representative strain of novel H1N1, was prepared. This virus was grown in Madin-Darby canine kidney (MDCK) cells and had a titer of 107.5 50% tissue culture infectious dose (TCID50/mL). Each virus dilution was tested in duplicate using the three RIDTs. Only specimens that tested positive for both test runs were considered positive. Limits of detection were measured as Ct values for the three RIDTs. The limit of detection of MDCK-grown A/California/4/2009 was the same for QuickVue A+B and Directigen EZ Flu A+B, but BinaxNOW Influenza A&B was 10-fold higher (10-2 versus 10-3) (Table 2).

Editorial Note:

The sensitivity of RIDTs to detect seasonal influenza viruses compared with virus isolation or rRT-PCR varies among commercial kits and has been shown to be low in some reports (3--5). In this evaluation, the sensitivity of three RIDTs to detect novel influenza A (H1N1) viral antigen in clinical specimens ranged from 40% to 69% and declined substantially with lower viral titers (as determined by Ct values). These findings are compatible with other recent studies, which reported that the sensitivity of some RIDTs to detect novel influenza A (H1N1) in clinical specimens ranged from 10% to 51% (6,7). Overall, the findings in this report demonstrate that these RIDTs are capable of detecting novel influenza A (H1N1) in respiratory specimens, but that many infections will be missed, especially in specimens with low viral titers.

RIDTs do not distinguish among influenza A virus subtypes, and RIDT sensitivity might vary by subtype of influenza A (4,6,8). Therefore, when using a positive RIDT result to help determine the appropriate course of clinical treatment or other action, the result should always be interpreted in the context of currently circulating strains. Conversely, as indicated by the results of this and other studies, a negative RIDT result should not be interpreted as indicating the absence of infection. In this analysis, the sensitivity of all three assays evaluated declined as the viral titer in the specimen decreased. The amount of virus found in respiratory specimens can be affected by timing of the specimen collection; viral titers are highest in the first 3 days of illness. Other factors that can affect the amount of virus in the specimen include age (e.g., children generally shed more virus and for longer periods than adults), type of specimen collected, and transportation and storage of the specimen before testing. Testing with rRT-PCR or virus isolation should be performed if a more definitive determination of the presence of influenza virus is required. In the titered cultured virus results presented in this report, all three RIDTs detected the cultured novel H1N1 influenza A/California/4/2009 virus with a lower limit of detection between 104.5 and 105.5 TCID50, slightly higher TCID50 levels than for detection of seasonal influenza viruses. These findings are consistent with the analytical sensitivities of RIDTs to detect novel influenza A (H1N1) virus described in one report (9), but higher than those described in another report (10).

The findings in this report are subject to at least three limitations. First, relatively few clinical specimens were tested for each RIDT across the range of Ct values, limiting the ability to compare results between different RIDTs, particularly for seasonal influenza A (H1N1). Second, clinical specimens were not tested immediately after collection but were stored and shipped to CDC under varying conditions. The clinical materials used in this evaluation were prepared and shipped in different (often unknown) transport media that might not be optimal for some of the RIDTs. Finally, the data used to estimate virus load in clinical materials obtained by comparing with different dilutions of influenza A/California/4/2009 grown in MDCK cells should be viewed with caution, because Ct limit of detection values for cultured viruses can vary with the virus strain, its passage history, and the substrate used for propagation (e.g., MDCK cells or chicken embryos). Optimizing specimen collection, transportation, and testing practices to ensure that specimens have the highest amount of virus possible would be expected to increase the likelihood of detecting influenza virus, when present, using RIDTs and other diagnostic tests.

The results described in this report should be viewed as preliminary. More data are needed on the clinical performance of all RIDTs to detect novel influenza A (H1N1) virus in different respiratory specimens. Because of the limitations of RIDTs and until additional data are available, all results from RIDTs, both positive and negative, when used for clinical decision-making in a patient with suspected novel influenza A (H1N1) virus infection, should be interpreted in the context of circulating influenza virus strains in the patient's community, level of clinical suspicion, severity of illness, and risk for complications. Additional CDC guidance on interpretation of RIDTs for testing of patients with suspected novel influenza A (H1N1) virus infection is available at http://www.cdc.gov/h1n1flu/guidance/rapid_testing.htm.

Acknowledgments

This report is based, in part, on contributions from national and international laboratories participating in the Global Influenza Surveillance Network.

** Only 43 of the 45 specimens positive for novel influenza A (H1N1) by rRT-PCR were tested using this RIDT.

TABLE 1. Comparison of the number of positive influenza A test results from three RIDTs* with the number of positive results from rRT-PCR† assay, by influenza A type and cycle threshold (Ct) interval --- United States, 2009

RIDT

Influenza A virus type

No. of specimens positive by RIDT/

No. positive by rRT-PCR

Total no. of specimens positive by RIDT/

Total no. positive by rRT-PCR

(%)

Ct interval§

(<20)

(20 to <25)

(25--30)

(>30)

BinaxNOW Influenza A&B

Novel H1N1

8/9

7/17

2/13

1/6

18/45

(40)

Seasonal H1N1

---¶

2/3

1/2

---

3/5

(60)

Seasonal H3N2

---

10/10

2/4

0/1

12/15

(80)

Directigen EZ Flu A+B

Novel H1N1

8/9

10/16

2/12

1/6

21/43**

(49)

Seasonal H1N1

---

2/2

1/2

---

3/4**

(75)

Seasonal H3N2

---

8/8

2/3

0/1

10/12**

(83)

QuickVue A+B

Novel H1N1

9/9

13/17

6/13

3/6

31/45

(69)

Seasonal H1N1

---

2/3

2/2

---

4/5

(80)

Seasonal H3N2

---

10/10

2/4

0/1

12/15

(80)

* Rapid influenza A diagnostic tests.

† Real-time reverse transcription--polymerase chain reaction.

§ A Ct value of 37 or lower is considered a positive rRT-PCR result.

¶ No data available.

** For this RIDT, insufficient material was available to test two specimens that were rRT-PCR positive for novel H1N1, one for seasonal H1N1, and three for seasonal H3N2

† Ct (cycle threshold) values reported as an average of three reactions each of duplicate dilution series.

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